Abstract Opioid use disorder (OUD) and HIV infection are syndemic conditions that independently and synergistically lead to central nervous system (CNS) dysfunction in tens of millions of people globally. However, the cellular circuits altered by OUD and HIV, and their combination, remain elusive. Further, the identities of cell types within the brain that can harbor HIV infection remain controversial. To address this key vexing question of HIV location within the brain and the effects of HIV and OUD on the brain, comprehensive tissue characterization at the single-cell level is needed to identify novel rare cell types, enriched or depleted cellular populations, and cellular circuits tied to pathogenesis. We propose to employ state-of-the-art methodologies in a center at Yale devoted to generating data on Single Cell Opioid Responses in the Context of HIV Discovery (SCORCH), Y-SCORCH. The center assembles a team of investigators at Yale with leading expertise in neurogenomics, HIV biology, neuroscience of addiction, single-cell analytics and consortium science, and a record of existing collaborations. Our TISSUE Component includes plans to sample 20 brains from four donor groups: controls, HIV (HIV+), HIV with OUD (HIV+OUD+), and OUD without HIV (OUD+). For each brain we will study 4 regions (prefrontal cortex, ventral striatum, insular cortex, and amygdala), representing disease-relevant areas for OUD and HIV. Our ASSAY Component will carry out single nucleus RNA sequencing (snRNA-seq) for 5,000-20,000 cells/sample, single-nucleus ATAC-seq (scATAC-seq), and spatial transcriptomics to generate transcriptomic, epigenetic, and spatial atlases for each donor type and each region. In parallel, we will detect HIV transcripts in the HIV+ groups. Our DATA Deposition & Analysis Component will assemble data standards, facilitate dissemination, and integrate our data with existing brain atlases. It will develop pipelines for high-throughput data analysis, including for single nucleus transciptomes, detection of HIV transcripts and for scATAC-seq data, and develop innovative analysis methods. Our Prioritization & Functional VALIDATION Component proposes a process of identification of brain regions and donor types that best differentiate disease states, and describes experiments to validate the findings generated by transcriptomic and epigenetic analyses. Finally, our Research MANAGEMENT Component provides a framework for data sharing with the SCORCH Data Center and broader Consortium and ensures timely progress in achieving our milestones which include generating `omics data from 640 assays. Our Specific Aims are to: (1) Establish a workflow from tissue samples to single-cell data deposited in the data center, (2) Run the combined experimental and computational workflows on procured specimens, and (3) Follow up on large-scale data production with validation and further analysis. Y-SCORCH has established expertise in all approaches necessary to successfully create single-nucleus transcriptomic data to provide a scaffold for future discovery to inform pathophysiological understanding of CNS effects of OUD and HIV.